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Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction.

ABSTRACT: A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP+ and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically-modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin-peroxide intermediate in a catalytically-competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multi-cofactor active site, which has implications for enzyme design based on cofactor engineering approaches.

SUBMITTER: Martinoli C

PROVIDER: S-EPMC3893305 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction.

Martinoli Christian C Dudek Hanna M HM Orru Roberto R Edmondson Dale E DE Fraaije Marco W MW Mattevi Andrea A

ACS catalysis 20130101 12

A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP<sup>+</sup> and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically-modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center ...[more]

PMID: 24443704

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Project description:BackgroundThe large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.ResultsHerein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.ConclusionsResults show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

Dataset Information

Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction.

Publications

Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction.

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